In vivo assay system for screening and validation of drugs and other substances

ABSTRACT

The present invention relates to the development of an in vivo assay system, useful for developing therapeutic options by testing the efficacy of drugs and other substances, said system constituting a novel, fast, and inexpensive in vivo assay system wherein, transgenic Drosophila harbors human Adenomatous Polyposis Coli gene, a negative regulator of β-catenin.

FIELD OF THE INVENTION

[0001] The present invention relates to the development of an in vivoassay system. The said system is useful for developing therapeuticoptions. The drugs and other substances are tested for their efficacyusing said system. The said system constitutes a novel, fast, andinexpensive in vivo assay system. The said system is consisting oftransgenic Drosophila harboring human Adenomatous Polyposis Coli gene, anegative regulator of β-catenin.

BACKGROUND AND PRIOR ART REFERENCES OF THE INVENTION

[0002] Studies over the decades have identified a number of genes thatare associated with the transformation of normal cells to cancer cellsand the progression of benign tumors to malignant tumors.

[0003] For prevention, one would like to target drugs to genes that arealtered early in the process, whereas gene that are altered at variousstages of cancer progression form targets for therapeutic drugselection.¹

[0004] Identification of suitable targets for drug screening andvalidation of a potential drug as specific modifier of a given targetrequires simple model systems amenable to various kinds of genetic andin vivo biochemical analyses.

[0005] β-Catenin, a transducer of Wnt signaling, is a potentialoncogene, activation of which is the cause of many different kinds ofcancer, such as colon cancer, melanomas, ovarian cancer,medulloblastoma, prostate cancer, uterine cancer etc. In the absence ofWnt signaling, free cytoplasmic β-catenin is subjected toubiquitin-mediated protein degradation.

[0006] According to the current model of β-catenin regulation, APC(protein product of human Adenomatous Polyposis Coli gene) forms acomplex with β-catenin, which is then recruited, by Axin/GSK-3β complex.This complex formation allows β-catenin to be phosphorylated by GSK-3βand the phosphorylated β-catenin is the target of ubiquitin-mediateddegradation pathway.

[0007] Stabilization of β-catenin is caused by gain-of-functionmutations in wnt-1 proto-oncogene or loss-of-function mutations intumor-suppressor genes APC (U.S. Pat. No. 5,736,389) or Axin. Directoncogenic activation of β-catenin is also reported, which is caused byspecific mutations in β-catenin that prevent its phosphorylation byGSK-3β.

[0008] Method of producing transgenic Typhoid fly is discussed for MUSCADOMESTICA (Russian Patent No.-2137362). Also, APC function, especiallythe down regulation of β-catenin appears to be conserved betweenDrosophila and humans (Hayashi et al., 1997; Ahmed et al., 1998).

[0009] In addition, Drosophila homologue of APC, dAPC can mediatedegradation of β-catenin in colon epithelial cells (Hayashi et al.,1997).

[0010] Study is been done on an in situ insect brain tissue assay,particularly suited for Dipteran brain tissue. The assay centres on theobservation by the present inventors that calcium levels in the brain ofDrosophila appear to oscillate when detected using a system employingtransgenic apoaequorin to measure intracellular levels of calcium in thebrain tissue.

[0011] Extensive biochemical analysis of APC protein, mostly in vitroand cell culture studies, has resulted in the identification of bindingsites for a number of proteins. Most important of them, in the contextof tumorigenesis, is the central region that harbors β-catenin bindingdomains (Polakis, 1997).

[0012] Cell lines with mutations in the APC gene show enhanced levels ofβ-catenin, which suggest that APC negatively regulates the cytoplasmiclevels of β-catenin, increase in which may affect Wnt/Wg signalingand/or cell adhesion. It has been shown that in the absence of theWnt/Wg signaling, the cytoplasmic β-catenin is phosphorylated andtargeted for ubiquitin-proteosome mediated degradation (Polakis, 1997;Polakis, 1999). As β-catenin does not directly interact with GSK-3 β,the formation of a complex consisting of GSK-3 β, Axin and APCfacilitates its phosphorylation and subsequent degradation.

[0013] Recently, it has been hypothesized that APC is involved in thetransportation of unphosphorylated β-catenin to the degradation complexlocated near the plasma membrane (Bienz, 1999).

[0014] Phenotypes have been reported for loss of wg function duringpupal development (Shirras and Couso, 1996; Kopp et al., 1999).

[0015] Wg and Decapentaplegic (Dpp) are known to repress each other'sexpression during leg development (Morimura et al., 1996; Brook andCohen, 1996). The mutually antagonistic interactions between Wg and Dppensure the maintenance of their expression in spatially restricteddomains. The combined action of these two genes activates Distal-less(Dll) expression in the central region of the leg disc overlapping theanterior-posterior compartment boundary (Diaz-Benjumea et al., 1994).Dll is required for the proper specification of leg structures along theproximal-distal axis (Gorfinkiel et al., 1997; Campbell and Tomlinson,1998).

[0016] Altered expression patterns of Wg and Dpp (Brook and Cohen, 1996)and phenotypes induced by loss of Wg (Held et al., 1997) are observed.Also, morphogenetic furrow progression (Ma et al., 1993, Treisman andRubin, 1995) is documented. Dpp causing inhibition of morphogeneticfurrow progression, which in turn leads to loss of ommatidia (Treismanand Rubin, 1995).

[0017] Similar leg and eye phenotypes were also induced by theintra-cellular domain of Drosophila E-cadherin (UAS-cad^(i5)); ( Sansonet al., 1996). Human APC could alleviate the effects of higher levels ofβ-catenin/Arm, when co-expressed with Flu-ΔArm (Zecca et al., 1996).

[0018] Increase in eye pigmentation and absence of pseudopupil is anindication of degeneration of retinal neurons (Ahmed et al. 1998).

[0019] Recently, it has been reported that all of the C-terminal domainsof APC are dispensable in mouse. A mouse model homozygous for APC1638T,expressing a truncated protein with ability to bind β-catenin, is viableand tumor-free (Smits et al., 1999). This was supported by theobservation that expression of the β-catenin binding domain of human APCalone was enough to down regulate β-catenin activity in colon cancercell lines (Shih et al., 2000).

[0020] All the amino-acids (Arginine 386, Lysine 345 and Tryptophan 383of human β-catenin; (Von Kries et al. 2000) in β-catenin that areassociated with APC binding are conserved between Drosophila and humanβ-catenins (See “SEQ comparison 1” below).

[0021] Genetic studies described here on the in vivo function of APChave direct relevance in understanding the genesis of certain cancertypes. For example, particular melanoma cell line, namely 888 met,carries a single amino acid mutation, Ser³⁷ to Phe³⁷ substitution, inthe N-terminal region of β-catenin.

[0022] Over-expression of APC in 888 mel cells does not down regulateβ-catenin levels, suggesting that the mutant β-catenin is resistant todegradation. Interestingly, the mutant β-catenin is found accumulated onthe APC protein and the two are localized near the plasma membrane(Rubinfeld et al., 1997).

[0023] In addition, it has been observed that even the over-expressedAPC immuno-precipitates with β-catenin from 888 mel cells. Thisobservation has been the basis for some speculation on the roles ofβ-catenin, one of them being down regulation of an unknown growthcontrol function of APC (Polakis, 1997).

[0024] In the background of above results, it is plausible that in 888mel cells both endogenous and over-expressed APC bind the mutantβ-catenin with the same efficiency with which they bind wild typeβ-catenin. Since the mutant β-catenin is resistant to degradation due tothe Ser³⁷ to Phe³⁷ substitution, the mutant β-catenin sequesters APC byinhibiting its reuse: all the endogenous APC would remain bound to themutant β-catenin.

[0025] Constitutive activation of Wnt/Wg signaling would then beachieved by the excess β-catenin. (both wildtype and mutant forms).Prediction from this model would be, although over-expressed APC doesnot down regulate β-catenin levels, it would still inhibit β-cateninactivity. 888 mel cells also show changed cell-adhesion properties(Polakis, 1997).

[0026] Since β-catenin bound APC resides in the extensions of the plasmamembrane (Nathke et al., 1996; Rubinfeld, 1997, Polakis, 1997; Bienz,1999; this report), some amount of degradation-resistant β-catenin mayultimately reach the adhesion zones and thereby cause changes in thecell-adhesion properties. This can be attributed to the relativeaffinities of E-cadherin and APC to bind β-catenin/Arm: E-cadherin bindsto β-catenin/Arm with higher affinity than APC.

[0027] Recently, this classical genetic approach has been used toidentify genes that modulated the phenotypes induced by the ectopicallyexpressed human p53 in flies (Yamaguchi et al., 1999). UAS-hAPC/CBD wascrossed to ey-GAL4 in the presence of additional mutations in the genome(only the 3^(rd) chromosome mutations were tested). It was observed thata deletion in the polytene region 85F (uncovered by Df(3R)by62 and notby its overlapping deficiency Df(3R)by10) enhanced the eye phenotypesinduced by human APC to such an extent that many flies were totallyeyeless (FIG. 5; Table 1).

[0028] Twins (tws) is one of the many genes mapped to 85F region(http://flybase.bio.indiana.edu), which codes for the regulatorysub-unit of protein phosphatase 2A (PP2A). Biochemical experiments haveimplicated a role for PP2A in stabilizing β-catenin in the cytoplasm.

[0029] However, none of the three alleles of tws (P1568, tws⁵⁵ andtws⁶⁰) that were tested enhanced human APC-induced eye phenotypes.Alleles of other neighbouring genes such as Ras (Rase^(e1B)) and many ofthe available lethal P-insertions (P237, P1595, P1659 and P1783) mappingto 85F region (http://flybase.bio.indiana.edu) were also tried. Noneenhanced the human APC-induced eye phenotypes. This suggests thepossibility of a hitherto unknown component of Wg signaling pathway in85F region. Currently, EMS- and P-element induced mutations are beingused to identify the gene.

[0030] This gene is being characterized and shows enhancement ofphenotypes of certain Wg alleles such as Wg^(P) and Spd^(f1g), Spd^(hL2)(R Bajpai and LSS, unpublished observations).

[0031] APC anchors to different parts of the cell for efficienttransport of β-catenin from the cytoplasm to the plasma membrane(Neufeld and White, 1997; Yu et al., 1999; Bienz, 1999; McCartney andPeifer, 2000). Consistent with this, in the intestine, APC is present athigher levels in the crypt/villus boundary (wherein larger amounts ofβ-catenin is released from adhesion complexes; Nathke et al., 1996). Itis interesting to note here that invertebrate homologues of APC havemuch simpler secondary structure than their vertebrate counterparts

[0032] Wnt/Wg signaling stabilizes human APC/β-catenin/Armcomplex.(Papkoff et al., 1996).

[0033] It has been shown that β-catenin/Arm degradation complex,consisting of dAPC2 and Axin, is formed in the epithelial cells near theapicolateral adhesive zones (McCartney et al., 1999; Yu et al., 1999).Human APC is also mainly localized near the plasma membrane (Nathke etal., 1999). It has been proposed that APC-dependent anchoring of theβ-catenin/Arm degradation complex near the adhesion zones might enablecells to degrade free β-catenin/Arm more efficiently (Bienz, 1999

[0034] Due to N-terminal deletions UAS-S10 and Flu-ΔArm would not bephosphorylated by whatever the residual GSK-3β is present (Zecca et al,1996; Pai et al., 1997).

[0035] It has been reported earlier that over-expression of Wnt-1 (whichin turn inhibits GSK-3 β activity) in mouse mammary epithelial orpituitary cell lines is known to cause increased stabilization ofAPC/β-catenin complex (Papkoff et al., 1996). It has also been shownthat in human cell lines, APC efficiently binds degradation-resistantforms of β-catenin (Rubinfeld et al., 1996). Degradation of β-cateninmay help the cells to reuse APC protein (Bienz, 1999).

[0036] Attenuated Polyposis or nonpolyposis phenotypes are documented(Scott et al., 1996; Friedl et al., 1996).

[0037] Indomethacin is a non-steroid anti-inflammatory drug (NSAID) andaffects the transcription downstream of peroxisome proliferatesactivated receptor—type δ (PARδ); (He et al., 1999), a member of nuclearhormone receptors.

OBJECT OF THE PRESENT INVENTION

[0038] The main object of the present invention is to develop an assaysystem using regulated mis-expression of the human Adenomatous PolyposisColi (APC) gene in Drosophila.

[0039] Another object of the present invention is to use the said assaysystem to develop preventive and therapeutic drugs.

[0040] Yet another object of the invention is to use the said system todetermine the modulation and differential expression of the several APCinteracting genes.

[0041] Still another object of the present invention is to determine thenew target proteins interacting with β-catenin.

[0042] Still another object of the present invention is to understandthe kinetics of Wnt/Wg signal transduction in Drosophila.

[0043] Still another object of the present invention is to studybiochemical function of human APC function.

[0044] Still another object of the present invention is identifyadditional components of Drosophila Wnt/Wt signaling pathway.

SUMMARY OF THE PRESENT INVENTION

[0045] The present invention relates to the development of an in vivoassay system. The said system is useful for developing therapeuticoptions. The drugs and other substances are tested for their efficacyusing said system. The said system constitutes a novel, fast, andinexpensive in vivo assay system. The said system is consisting oftransgenic Drosophila harboring human Adenomatous Polyposis Coli gene, anegative regulator of β-catenin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0046] Accordingly the present invention relates to a transgenicDrosophila whose genome comprises the full-length human colon cancergene Adenomatous Polyposis Coli (APC) having SEQ ID NO. 1 wherein:

[0047] (a) said genomic alteration allows mis-expression of full-lengthhuman APC in flies in regulated manner,

[0048] (b) said mis-expression of the full-length human APC results indevelopmental abnormalities,

[0049] (c) said developmental abnormalities induced by themis-expression of full-length human APC in flies are similar to thoseexhibited by flies carrying mutations in Drosophila wingless gene, and

[0050] (d) to use the same as an assay system for screening andvalidating efficacy of drugs.

[0051] In an embodiment of the present invention the transgenicDrosophila genome includes β-catenin binding domain comprising ofamino-acids from 959 to 1870 of SEQ ID NO. 2 from the full length humanAPC gene of SEQ ID NO.1, and this engineered disruption of human APCcomprises only the five of the seven β-catenin binding domains wherein:

[0052] (a) said genomic alteration allows mis-expression of a truncatedversion of human APC in flies in a regulated manner,

[0053] (b) said mis-expression of the said gene construct results in thedevelopmental abnormalities,

[0054] (c) said developmental abnormalities induced by themis-expression of the said gene construct in flies is similar to thoseexhibited by flies carrying mutations in Drosophila wingless gene,

[0055] (d) said mis-expression of the said novel construct in regulatedmanner results in a more severe developmental phenotype, and

[0056] (e) to use the same as an assay system for screening andvalidating efficacy of drugs.

[0057] In another embodiment of the present invention, transgenicDrosophila has the N terminal domain of APC with amino acids from 1 to767 having SEQ ID NO. 3, from the full length human APC gene of SEQ IDNO.1,wherein:

[0058] (a) said genomic alteration allows mis-expression of human APC inflies in a regulated manner,

[0059] (b) said mis-expression of the said novel construct in aregulated manner resulting in severe abnormalities in fly developmentduring metamorphosis, and

[0060] (c) to use the same as an assay system for screening andvalidating efficacy of drugs.

[0061] In yet another embodiment of the present invention, a method forselecting a compound for pharmacological activity, which potentiallyinhibits or enhances the developmental abnormalities induced by theexpression of full length and protein domains of human APC inDrosophila, said method comprising:

[0062] (a) providing the above-mentioned first, second, and thirdtransgenic fly wherein said flies have said developmental abnormalities,

[0063] (b) administering the said compounds to the said transgenicDrosophila at different concentrations, and

[0064] (c) screening for the changes in the severity of the phenotype.

[0065] In still another embodiment of the present invention, a method ofdetermining various Drosophila proteins interacting with full-length andprotein domains human APC protein wherein, said method comprising:

[0066] (a) providing the above-mentioned first, second, and thirdtransgenic fly, wherein said flies have said developmentalabnormalities,

[0067] (b) crossing the said transgenic flies individually to a set ofDrosophila strains each of which carries mutation in a different gene orset of genes, and

[0068] (c) Screening for the change in the severity of the phenotype.

[0069] In still another embodiment of the present invention, a methodfor determining the modulation and differential expression of genesfollowing the mis-expression of full-length and its protein domainshuman APC in Drosophila wherein, said method comprising:

[0070] (a) providing the above-mentioned transgenic Drosophila wherein,the flies have developmental abnormalities,

[0071] (b) screening for differential gene expression using differentialdisplay-RT PCR or microarray techniques, and

[0072] (c) identifying genes that are differentially regulated onexpression of human APC.

[0073] In still another embodiment of the present invention, a methodfor determining the modulation and differential expression of proteinsfollowing the mis-expression of full-length and its protein domain humanAPC in Drosophila wherein, said method comprising:

[0074] (a) providing the above-mentioned transgenic Drosophila the flieshave developmental abnormalities,

[0075] (b) identifying differential gene expression and proteinmodifications using proteomics techniques, and

[0076] (c) identifying gene products that are differentially regulatedon expression of human APC.

[0077] In still another embodiment of the present invention, a method tostudy Wnt/Wg signaling in Drosophila said method comprising;

[0078] (a) providing the above-mentioned transgenic Drosophila,

[0079] (b) crossing these transgenic flies to a number of GAL4 driversto induce targeted expression of said constructs in various tissues andat different developmental stages, and

[0080] (c) examining developmental abnormalities.

[0081] In still another embodiment of the present invention, methodswherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC to study mechanism ofvarious developmental processes such as wing, leg, eye, antennae, andadult cuticle development.

[0082] In still another embodiment of the present invention, a methodwherein, screening and validating efficacy of preventive and therapeuticdrugs following APC gene mis-expression.

[0083] In still another embodiment of the present invention, a methodwherein, human APC pathway is identified using drug selected from agroup of compounds comprising anti inflammatory, Analgesics,Antipyretics, and Antineoplastics.

[0084] In still another embodiment of the present invention, a methodwherein, concentration of said drugs ranging between 50 to 500 μg/ml offly food.

[0085] In still another embodiment of the present invention, methodswherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC which has advantages tostudy the Drosophila Wnt/Wg signaling pathway.

[0086] In still another embodiment of the present invention, a methodwherein, studying the kinetics of Wnt/Wg signaling during variousdevelopmental stages and in different tissues.

[0087] In still another embodiment of the present invention, a methodwherein, new target proteins interacting with β-catenin are identified.

[0088] In still another embodiment of the present invention, a methodwherein, genes interacting with APC are identified.

[0089] In still another embodiment of the present invention, methodswherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC to study biochemicalfunction of human APC function.

[0090] In still another embodiment of the present invention, methodswherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC to identify additionalcomponents of Drosophila Wnt/Wg signaling pathway.

[0091] In still another embodiment of the present invention, atransgenic Drosophila whose genome comprises the full-length human coloncancer gene Adenomatoits Polyposis Coli (APC) having SEQ ID NO.1wherein:

[0092] (a) said genomic alteration allows mis-expression of full-lengthhuman APC in flies in regulated manner,

[0093] (b) said mis-expression of the full-length human APC results indevelopmental abnormalities,

[0094] (c) said developmental abnormalities induced by themis-expression of full-length human APC in flies are similar to thoseexhibited by flies carrying mutations in Drosophila wingless gene, and

[0095] (d) to use the same as an assay system for screening andvalidating efficacy of anti-cancer drugs.

[0096] In still another embodiment of the present invention, thetransgenic Drosophila wherein, its genome includes β-catenin bindingdomain comprising of amino-acids from 959 to 1870 of SEQ ID NO. 2 fromthe full length human APC gene of SEQ ID NO.1, and this engineereddisruption of human APC comprises only the five of the seven β-cateninbinding domains wherein:

[0097] (a) said genomic alteration allows mis-expression of a truncatedversion of human APC in flies in a regulated manner,

[0098] (b) the mis-expression of the said gene construct results in thedevelopmental abnormalities,

[0099] (c) the developmental abnormalities induced by the mis-expressionof the said gene construct in flies is similar to those exhibited byflies carrying mutations in Drosophila wingless gene,

[0100] (d) mis-expression of the said novel construct in regulatedmanner results in a more severe developmental phenotype, and

[0101] (e) to use the same as an assay system for screening andvalidating efficacy of anti-cancer drugs.

[0102] In still another embodiment of the present invention, transgenicDrosophila wherein, the N terminal domain of APC with amino acids from 1to 767 having SEQ ID NO. 3, from the full length human APC gene of SEQID NO.1 wherein:

[0103] (a) the said genomic alteration allows mis-expression of humanAPC in flies in a regulated manner,

[0104] (b) the mis-expression of the said novel construct in a regulatedmanner resulting in severe abnormalities in fly development duringmetamorphosis, and

[0105] (c) to use the same as an assay system for screening andvalidating efficacy of anti-cancer drugs.

[0106] In still another embodiment of the present invention, a methodfor selecting a compound for anti-cancer activity, which potentiallyinhibits or enhances the developmental abnormalities induced by theexpression of full length and protein domains of human APC inDrosophila, said method comprising:

[0107] (a) providing the first, second, and third transgenic fly,wherein said flies have said developmental abnormalities,

[0108] (b) administering the said compounds to the said transgenicDrosophila at different concentrations, and

[0109] (c) screening for the change in the severity of the phenotype.

[0110] In still another embodiment of the present invention, a methodwherein, screening and validating efficacy of anti-cancer drugsfollowing APC gene mis-expression.

[0111] In still another embodiment of the present invention, a methodwherein, human APC pathway is identified using drugs selected from agroup of anti-cancer compounds comprising anti inflammatory, Analgesics,Antipyretics, and Antineoplastics.

[0112] In still another embodiment of the present invention, a methodwherein, concentration of said anti-cancer drugs ranging between 50 to500 μg/ml of fly food.

[0113] In yet another embodiment of the present invention, a transgenicnon-human animal expressing human colon cancer gene, Adenomatouspolyposis Coli (APC). Applicants have generated transgenic flies thatexpress human APC, an important tumor suppressor gene, and mutations inwhich cause both familial and sporadic colorectal cancers.

[0114] In still another embodiment of the present invention, transgenicDrosophila expressing either full length or truncated forms of human APCprotein and methods for using it as an assay system for identificationand development of preventive as well as therapeutic drugs againstcancer and other disease conditions.

[0115] In still another embodiment of the present invention, consistentwith its biochemical properties, targeted expression of eitherfull-length APC or its truncated form negatively regulate the functionof the β-catenin homologue, Armadillo (Arm). This in turn results in theinhibition of Wnt/Wg signaling during fly development and give rise tophenotypes similar to those associated with loss of Wnt/Wg signaling inDrosophila.

[0116] In still another embodiment of the present invention, thetransgenic fly of the invention can be used to study genetic andbiochemical pathways associated with cotorectal and other cancers.

[0117] In still another embodiment of the present invention, thephenotypes generated in the fly due to the mis-expression of human APCwould constitute a novel, fast and less expensive in vivo assay systemfor the identification of targets for drug screening and validation ofvast repertoire of drugs against cancer and other disease conditions.

LIST OF THE ACCOMPANYING DRAWINGS

[0118]FIG. 1 shows human APC constructs used for generating transgenicflies and phenotypes induced by their expression in Drosophila.

[0119]FIG. 2 shows human APC suppression phenotypes, induced by elevatedlevels of β-catenin/Arm and rescues phenotypes associated with mutantdAPC.

[0120]FIG. 3 shows human APC induced changes in the levels ofβ-catenin/Arm in different cell types.

[0121]FIG. 4 shows effect of GSK-3 β/Sgg activity on the interactionbetween human APC and β-catenin/Arm.

[0122]FIG. 5. shows enhancement of human APC induced eye phenotypes inDrosophila.

[0123] Table 1 Distribution of eye phenotypes induced by hAPC/CBD indifferent genetic backgrounds and in the presence of various compoundsand other anti-cancer drugs. Numbers are from a representativeexperiment. Number in parenthesis in the last column indicates totalnumber of the flies of right genotype. Phenotypic ranks Genotypes 1-56-10 11-15 16-20 Total Ey-GAL4; hAPC/CBD 13 5 — — 18(49) Ey-AL4;hAPC/CBD/ 10 7 12  9 38(86) Df(3R)by⁶² Ey-GAL4; hAPC/CBD/  7 6 6 524(63) 1(3L)67E2 Ey-GAL4; hAPC/CBD in the  9 4 1 — 14(47) presence of5-fluorouracil at 50 micrograms/ml fly food. Ey-GAL4; hAPC/CBD in the 128 16  11  47(121) presence of Indomethacin at 200 microgram/ml fly food.Ey-GAL4; hAPC/CBD in the 13 4 2 — 19(61) presence of aspirin at 200micrograms/ml fly food. Ey-GAL4; hAPC/CBD in the 11 7 1 2 21(59)presence of Pyroxicam at 500 micrograms/ml fly food.

[0124] In an embodiment of the present invention, transgenic flies aregenerated for the full-length human APC (hAPC/FL) as well as for theβ-catenin binding domain (hAPC/CBD) and the N-terminal domain (hAPC/NBD)(FIG. 1A; Example 1).

[0125] In another embodiment of the present invention, GAL4-UAS systemis employed for targeted expression of human APC constructs in differenttissues during development and in a number of genetic backgrounds.

[0126] In yet another embodiment of the present invention, alternativeexpression systems such as those, which employ heat-shock ortissue-specific promoters are also available which can also be used forexpression of genes of interest. These systems may also be effective ingiving phenotype with human APC.

[0127] In still another embodiment of the present invention, bothfull-length and the β-catenin binding domain give comparable phenotypes,although levels of expression of full-length protein are lower thanthose of the β-catenin binding domain.

[0128] In still another embodiment of the present invention, effects ofhAPC/FL expression are less severe than hAPC/CBD. Wherever both hAPC/FLand hAPC/CBD give comparable phenotypes, the term human APC is used torefer to them together. Mis-expression of hAPC/NBD resulted in severedefects in metamorphosis and head involution.

[0129] In still another embodiment of the present invention, expressionof human APC in flies was confirmed by both RNA in situ (data not shown)and antibody staining (FIGS. 3B, F). It was first ensured that behaviorof APC in flies is consistent with its biochemical role in human.

[0130] In an embodiment of the present invention, to determine if humanAPC interferes with Wingless (Wnt/Wg) signaling in flies, its expressionis targeted to Wg-dependent developmental pathways.

[0131] In another embodiment of the present invention, UAS-hAPC/FL andUAS-hAPC/CBD are mis-expressed in different tissues such as epidermis,mesoderm, central and peripheral nervous systems and endodern and atdifferent developmental time stages with the help of a number oftissue-specific GAL4-driver lines.

[0132] In yet another embodiment of the present invention, both theabove-mentioned constructs induce phenotypes in adult legs, wings,antennae and eyes. In addition, the full-length construct, whenexpressed with ptc-GAL4 driver, affected adult epidermal development.The dorsal tergites are not properly developed and were devoid ofpigmentation (FIG. 3B).

[0133] In still another embodiment of the present invention, expressionof human APC in leg discs using ptc-GAL4 resulted in ectopic expressionof Dpp (FIG. 1D), suggesting negative regulation of Wnt/Wg signaling.

[0134] In still another embodiment of the present invention, ectopicDistal-less (Dll) expression is also seen (FIG. 1F), which reflectsaltered expression patterns of Wg and Dpp.

[0135] In still another embodiment of the present invention, adult fliesexpressing human APC exhibited leg phenotypes such as leg duplications(FIGS. 1H-J) that are similar to the phenotypes induced by loss of Wg.

[0136] In still another embodiment of the present invention, indeveloping eye imaginal discs too, Wg negatively interact with Dpp toregulate the morphogenetic furrow progression. Gain of Dpp or loss of Wgresults in the activation of retinal differentiation along dorsal andventral margins. This leads to inappropriate patterning of ommatidia,leading to either ectopic ommatidia in the anterior head capsule or insevere cases, loss of ommatidia.

[0137] In still another embodiment of the present invention, expressionof human APC in the eye disc using ey-GAL4 resulted in ectopic ommatidiaresulting in the protrusion of the eye over the anterior head capsule(FIGS. 1K-M) The phenotypes are identical to those induced by theover-expression of Dpp or activated form of its receptor Thick-vein,further suggesting down regulation of Wnt/Wg signaling by human APC.

[0138] In still another embodiment of the present invention, inaddition, human APC-induced phenotypes in many other appendages/tissuessuch as wing, antennae and adult cuticle all of which resemblephenotypes caused by reduced Wnt/Wg signaling. These observations revealthat human APC expression induced specific phenotypes, which mimic lossof Wg signaling.

[0139] In an embodiment of the present invention, similar leg and eyephenotypes are also induced by the intra-cellular domain of DrosophilaE-cadherin (U; AS-cad^(i5)), which is known to deplete the cytoplasmicpool of Armnadillo (β-catenin/Arm).

[0140] In another embodiment of the present invention, since APC is aknown β-catenin binding protein, it is hypothesized that humanAPC-induced phenotypes are also mediated by the negative regulation ofβ-catenin/Arm. This is supported by the enhanced leg phenotypes (oftenleading to total loss of appendage development) induced by theexpression of human APC using ptc-GAL4 in flies heterozygous for a nullmutation in arm.

[0141] In yet another embodiment of the present invention, in addition,human APC could alleviate the effects of higher levels of β-catenin/Arm,when co-expressed with Flu-ΔArm, a stable form of β-catenin/Arm (FIG.2C).

[0142] In still another embodiment of the present invention, finally,possibility of suppression of human APC-induced phenotypes by theover-expression of β-catenin/Arm is examined. Enhanced Wg activity indeveloping eye imaginal discs down-regulates Dpp causing inhibition ofmorphogenetic furrow progression, which in turn leads to loss ofommatidia. Consistent with this, mis-expression of Flu-ΔArm usingey-GAL4 result in severe loss of ommatidia. Although hAPC/CBD (FIGS.1K-M) and Flu-ΔArm (FIG. 2E) induce eye abnormalities when expressedindividually using ey-GAL4, flies expressing both had normal eyes (FIG.2F).

[0143] In still another embodiment of the present invention, thissuggests that, at least in the eye, human APC-induced phenotypes areentirely due to its interaction with β-catenin/Arm.

[0144] In still another embodiment of the present invention, theseobservations also suggest that human APC expression in the heterologoussystem does not lead to any dominant-negative effect.

[0145] In still another embodiment of the present invention, applicantsobserve that the down regulation of β-catenin/Arm activity in the abovementioned experiments is not a mere reflection of the presence ofoverwhelming amount of human APC, since human APC could also alleviatedominant phenotypes caused by the over-expression of β-catenin/Arm.

[0146] In still another embodiment of the present invention, to furthervalidate genetic studies on hAPC in this heterologous system, it isfurther determined whether hAPC could be substituted for its Drosophilahomologue dAPC.

[0147] In still another embodiment of the present invention, dAPC^(Q8),a viable allele of dAPC in which all surviving homozygous flies showincrease in eye pigmentation and absence of pseudopupil, an indicationof degeneration of retinal neurons (FIG. 2I) is used in the abovementioned experiment. hAPC/FL is expressed in dAPC^(Q8)/dAPC^(Q8)background with the help of 405-GAL4 driver, which is expressed in thedifferentiating retinal neurons (FIG. 2G). All the flies expressinghAPC/FL in dAPC^(Q8)/dAPC^(Q8) background show the presence ofpseudopupil and normal levels of eye pigmentation suggestingcomplementation of dAPC by hAPC/FL (FIG. 2J).

[0148] In still another embodiment of the present invention, applicantshave shown that human APC-induced phenotypes in flies are consistentwith the role of human APC as a negative regulator of β-catenin/Armactivity thereby, supporting the use of fly model system for geneticanalyses of human APC function.

[0149] In still another embodiment of the present invention, however,UAS-dAPC 1induce only very weak phenotypes when expressed using variousGAL4 lines and at different temperature conditions. Its ability tosuppress the phenotypes induced by over-expression of dominant forms ofβ-catein/Arm is also negligible. This is particularly intriguingconsidering the specificity of human APC-induced phenotypes in flies.One possible explanation is, human APC has higher affinity for Arm thandAPC1 or EAPC. This is supported by the fact that β-catenin is far morehighly conserved than APC across a number of species from Drosophila tohuman. For example all the amino-acids (Arginine 386, Lysine 345 andTryptophan 383 of human β-catenin; in β-catenin that are associated withAPC binding are conserved between Drosophila and human β-catenins (See“SEQ comparison 1” below).

[0150] In still another embodiment of the present invention, hAPC/CBDalone is enough to bind and transport β-catenin to degradation zone andto subject the same for degradation in the presence of GSK-3 β-activity.Interestingly, hAPC/CBD, which comprises only the five of the sevenβ-catenin binding domains result in far more severe developmentalphenotypes than hAPC/FL. This may reflect enhanced ability of human APCto bind β-catenin/Arm due to the engineered disruption of the protein.

[0151] In still another embodiment of the present invention, expressionof human APC result in stronger phenotypes indicates the uniqueness ofthis transgenic system to study genetic and biochemical pathways relatedto APC function.

[0152] In an embodiment of the present invention, the nature of humanAPC-induced adult phenotypes and its ability to suppress the effects ofdegradation-resistant Flu-ΔArm suggest down-regulation or sequestrationof β-catenin/Arm in human APC-expressing cells. Therefore, changes inthe levels of β-catenin/Arm following human APC expression weremonitored.

[0153] In another embodiment of the present invention, normally,β-catenin/Arm is present in all the cells, although its levels areslightly higher in cells where Wnt/Wg signaling is active (for example,in the wing disc D/V boundary; FIG. 3G). It is observed that a subset ofhuman APC-expressing cells exhibited enhanced levels of β-catenin/Arm(FIGS. 3D, H, J). In other cells, the levels of β-catenin/Arm arecomparable to the wildtype levels.

[0154] In yet another embodiment of the present invention, doublestaining for β-catenin/Arm and Wg, reveal that β-catenin/Arm isaccumulated in cells where human APC and endogenous Wg expressionoverlapped (FIG. 3I). Wnt/Wg signaling inhibits the degradation ofβ-catenin/Arm by down regulating the activities of GSK-3 β/Sgg and Axin(7). This results in the increased levels of cytoplasmic β-catenin/Arm.

[0155] In still another embodiment of the present invention,accumulation of β-catenin/Arm only in cells expressing both human APCand endogenous Wg suggests that (i) human APC can efficiently inhibitthe function of cytoplasmic β-catenin/Arm even in the cells whereShaggy/Zeste-White3 (GSK-3 β/Sgg) activity is down regulated by Wnt/Wgsignaling and (ii) human APC-induced phenotypes are due to thesequestration of β-catenin/Arm.

[0156] In still another embodiment of the present invention,immuno-precipitation and Western blot analyses does not show any markedincrease in the levels of β-catenin/Arm over the control. This is due tothe fact that human APC-mediated accumulation of β-catenin/Arm is seenonly in a small fraction of cells.

[0157] In still another embodiment of the present invention, since humanAPC can efficiently bind and sequester larger amounts of β-catenin/Armin cells responding to Wnt/Wg signaling, it is presumed that in othercells too it can bind β-catenin/Arm. It implies that in those cellseither human APC-bound form of β-catenin/Arm is recognized by theAxin-GSK-3 βSgg complex for degradation or no free cytoplasmicβ-catenin/Arm is available for human APC to bind. The latter possibilityis ruled out since ectopic expression of UAS-cad^(i5) resulted in theaccumulation of large and equal amounts of β-catenin/Arm in all thecells, irrespective of the presence or absence of Wnt/Wg signaling (FIG.3K).

[0158] In still another embodiment of the present invention, theregulation of β-catenin/Arm at the transcriptional level using arm-lacZconstruct is examined. There is no change in lacZ levels in response tohuman APC expression in different tissues. This suggests that humanAPC-induced increase in the levels of β-catenin/Arm protein (FIGS. 3D,H, J) is due to stabilization of human APC/β-catenin/Arm complex byWnt/Wg signaling.

[0159] In still another embodiment of the present invention, thedevelopment of a gain-of-function genetic model for human APC similar tothe one described here, has certain advantages to study Wnt/Wg signalingin Drosophila.

[0160] In still another embodiment of the present invention, the role ofβ-catenin/Arm in cell-adhesion has always been a limitation to generateloss-of-function clones of arm⁻ cells to study Wnt/Wg signaling duringpost-embryonic stages. Such clones are invariably cell-lethal due toloss of cell-adhesion and do not produce any data on Wnt/Wg signalingevents.

[0161] In still another embodiment of the present invention, it has beendemonstrated that mis-expression of human APC down-regulate Wnt/Wgsignaling by binding to cytoplasmic β-catenin/Arm, thus enabling one tostudy the kinetics of Wnt/Wg signaling during development.

[0162] In still another embodiment of the present invention, applicantshave examined the expression patterns of a number of limb patterninggenes (such as dpp, wg, Dll, dauschund, cut, etc.) by down-regulatingWnt/Wg signaling at different developmental stages and in differentregions of the developing imaginal discs.

[0163] In still another embodiment of the present invention, applicantshave also studied the requirement for Wnt/Wg signaling in differenttissues and developmental stages by inhibiting β-catenin/Arm activity.

[0164] In an embodiment of the present invention, in transgenic fliesexpressing human APC, both human APC and accumulated β-catenin/Arm arelocalized near the plasma membrane (FIGS. 3C1, F1, H1).

[0165] In another embodiment of the present invention, even in theabsence of any detectable accumulation of β-catenin/Arm i.e. in cellswhere GSK-3β/Sgg is active, much of the human APC is localized near theplasma membrane.

[0166] In yet another embodiment of the present invention, similarsub-cellular distribution of human APC in both kinds of cells, i.e.cells responding to Wnt/Wg signaling and otherwise, is an additionalevidence to show that human APC can not only bind Drosophila Arm, butalso can transport it to the plasma membrane for degradation.

[0167] In an embodiment of the present invention, the requirement ofGSK-3β/Sgg activity for human APC-mediated inhibition of β-catenin/Armfunction was examined. hAPC/CBD and the degradation-resistant form ofArm (UAS-S10 or Flu-ΔArm) are co-expressed in the wing disc D/V boundaryusing vg-GAL4.

[0168] In another embodiment of the present invention, in the wing discD/V boundary both Wg (FIG. 3G) and β-catenin/Arm (FIG. 3C) are maximallyexpressed and GSK3β/Sgg activity is expected to be minimum. In addition,due to N-terminal deletions UAS-S10 and Flu-ΔArm would not bephosphorylated by whatever the residual GSK-3β is present.

[0169] In yet another embodiment of the present invention, both hAPC/FLand hAPC/CBD suppress the phenotypes induced by Flu-ΔArm and UAS-S10(data not shown) with the same efficiency as it suppressed the ectopicβ-catenin/Arm activity in a non-Wg region (FIG. 2C).

[0170] In still another embodiment of the present invention, GSK-3β/Sggactivity is not essential for the binding of human APC to β-catenin/Armand the subsequent inhibition of β-catenin/Arm function.

[0171] In still another embodiment of the present invention, to furtherexamine the requirement of GSK-3β/Sgg activity for the humanAPC-mediated degradation of β-catenin/Arm, human APC and/or GSK-3β/Sggare expressed in wing imaginal discs using vg-GAL4 driver. Expression ofhAPC/CBD using vg-GAL4 resulted in the accumulation of large amount ofβ-catenin/Arm in the D/V boundary (FIG. 4B).

[0172] In still another embodiment of the present invention, whereas,over-expression of GSK-3 β/Sgg alone or together with hAPC/CBD result inreduced β-catenin/Arm levels in all the cells, which is more dramatic inthe D/V boundary (FIGS. 4C, D; compare with FIG. 3H).

[0173] In still another embodiment of the present invention, human APCbinds to β-catenin/Arm in all the cells, although β-catenin/Arm isdegraded only in cells where GSK-3β/Sgg is active. However, human APClevels are similar in all cells irrespective of the presence or absenceof GSK-3β/Sgg activity (FIG. 4E). The levels of human APC are notaltered even in the cells over-expressing GSK-3β/Sgg (FIG. 4F).

[0174] In still another embodiment of the present invention, thus, humanAPC is able to suppress with equal efficiency β-catenin/Arm-induceddominant phenotypes in the presence as well as in the absence ofGSK-3β/Sgg activity.

[0175] In still another embodiment of the present invention, theuncoupling of inhibition of β-catenin activity from its degradationsuggest that human APC inhibited Wnt/Wg signaling by sequesteringβ-catenin and not by the down regulation of its protein levels.

[0176] In still another embodiment of the present invention, theblockage of β-catenin degradation by Wnt signaling would affect therelease of free APC from the degradation complex. Thus, in cellscarrying gain-of-function mutations in Wnt or β-catenin, APC itself issequestered by β-catenin leading to accumulation of free β-catenin inthe cytoplasm, which would now be available to transduce Wnt signal.

[0177] In still another embodiment of the present invention, althoughthe final outcome would be similar to what happens in APC colon cancercells, the effects of loss-of-function mutations in APC are morepronounced in certain kinds of cells such as colonic crypts. Virtuallyall cells would be affected if the increased oncogenic activity ofβ-catenin is due to sequestration of APC.

[0178] In still another embodiment of the present invention, abolitionof Axin and APC interaction would inhibit the degradation of β-catenin,but would not affect the β-catenin binding ability of APC. Although thiswould lead to increase in β-catenin levels, the phenotype would be lesssevere when compared to that of APC⁻ cells. Indeed, colorectal cancerscaused by mutations in APC that delete only the Axin binding sites showeither attenuated polyposis or nonpolyposis phenotypes.

[0179] In addition, mutations in 67E2 (l(3)67E2 caused by the impreciseexcision of a P-element insertion) also showed enhanced eye-phenotypes,although less severely compared to Df(3R)by⁶² (Table 1).

[0180] In an embodiment of the present invention, transgenic fliesexpressing human APC are tested for their usefulness in screeningpotential drugs against colon cancer.

[0181] In another embodiment of the present invention, as pilotexperiments, some of the known anti-colon cancer drugs, for example,5-fluorouracil, pyroxicam, indomethacin, and aspirin are tested fortheir effect on Drosophila.

[0182] In yet another embodiment of the present invention, LD50 isdetermined for each of the chemicals tested. At concentrations LD50 andbelow, none of the drugs induce any morphological phenotypes in wildtypeflies. Thereafter, the effect of these drugs on transgenic fliesexpressing human APC in developing eyes (using ey-GAL4) is examined.

[0183] In still another embodiment of the present invention,Indomethacin enhances human APC-induced eye phenotypes to the extentsimilar to the enhancement shown by Df(3R)by⁶² (FIG. 5 and Table 1).

[0184] In still another embodiment of the present invention, the effectis concentration dependent, maximum being at 200 μg/ml of fly food (LD50being at 250 μg/ml of fly food).

[0185] In an embodiment of the present invention, the protein sequenceof PPARβ is compared to that of Drosophila proteins (known andpredicted). Eip75B, a transcription factor in ecdysone response pathway,shows 38% identity and 59% homology to PPARδ, (however, Eip75B is 1443amino acid long, whereas PPARδ is 441 amino acid long).

[0186] In another embodiment of the present invention, although Eip75Bis not known to modulate Wg signaling, these results show the potentialuse of transgenic flies in identifying targets (and thereby mode ofaction) of drugs that are already being used in addition to theirusefulness in identification of new target proteins.

[0187] In yet another embodiment of the present invention, applicantshave developed an assay system that enables relatively easy and fastidentification of suitable candidate genes for drug screening.

[0188] In still another embodiment of the present invention, this assaysystem allows the identification of molecular targets and biochemicalpathways modulated by currently used cancer drugs, whose mechanism ofaction is not known.

[0189] In still another embodiment of the present invention, applicantshave taken genetic approach to examine, in vivo, the interactionsbetween APC, β-catenin and other components of Wnt/wg signaling pathwayand the significance of the same in development and tumor formation.However, structure-function relationship studies of large multi-domainproteins, such as APC, require simple model systems amenable to variouskinds of genetic and in vivo biochemical analyses

[0190] In still another embodiment of the present invention, applicantshave generated flies transgenic for (i) the full-length human APC (ii)its N terminal fragment and (iii) for the fragment bearing only theβ-catenin binding domains. These transgenic flies are used to correlatebiochemical interactions amongst APC, β-catenin and GSK-3β to their invivo function

[0191] In still another embodiment of the present invention, consistentwith its biochemical properties, targeted expression of eitherfull-length APC or its β-catenin binding domain alone negativelyregulate the function of the β-catenin homologue, Armadillo (Arm) andthereby, inhibited Wnt/Wg signaling during fly development.

[0192] In still another embodiment of the present invention, althoughAPC is a large multi-domain protein, the β-catenin binding domain of APCalone is sufficient to functionally complement mutations in itsDrosophila homologue. APC inhibited Arm function even in the absence ofGSK-3β activity, although the latter is required to mediate thedegradation of Arm. Consistent with this, APC suppressed the phenotypesinduced by the over-expression of degradation-resistant forms of Arm.

[0193] In still another embodiment of the present invention,interestingly, transgenic construct carrying only β-catenin bindingdomain of human APC, which comprises only the five of the sevenβ-catenin binding domains resulted in far more severe developmentalphenotypes than full-length protein. This may reflect enhanced abilityof human APC to bind β-catenin/Arm due to the engineered disruption ofthe protein.

[0194] In still another embodiment of the present invention, moreover,the phenotypes induced by over-expression of human APC in Drosophila arefar more severe, although qualitatively similar, than those induced byDrosophila homologue of APC.

[0195] In still another embodiment of the present invention, thus, theobservation that mis-expression of human APC, particularly themis-expression of β-catenin binding domain alone, results in strongerphenotypes indicates the uniqueness of this transgenic system to studygenetic and biochemical pathways related to APC function.

[0196] In still another embodiment of the present invention, applicants'results show that transgenic flies expressing human APC is not only agood model system, but also a unique system for genetic studies on APCfunction.

[0197] In still another embodiment of the present invention, applicantshave identified two new loci in Drosophila, which act as positiveregulators of Wnt/Wg signaling.

[0198] In still another embodiment of the present invention, theusefulness of assays based on human APC-induced phenotypes in Drosophilafor the identification of targets for drug screening and validation ofpotential drugs against cancer and other diseases resulting due to highlevels of β-catenin is also demonstrated.

[0199] In still another embodiment of the present invention, applicantshave also confirmed that one of the currently used anti-cancer drugsspecifically enhanced human APC-induced phenotypes thereby demonstratingthat it is a specific inhibitor of Wnt signaling. This confirms theusefulness of the claimed genetic system for the screening of bioactivemolecules effective against cancer and other disease conditions.

[0200] In still another embodiment of the present invention, the humanAPC induced phenotypes in Drosophila can be employed for studying thefunction of the human APC protein and for developing therapeutics forthe prevention and treatment of cancer and other disease conditionscaused by high levels of β-catenin.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0201]FIG. 1 shows Human APC constructs used for generating transgenicflies and phenotypes induced by their expression in Drosophila. (A)Structure-function map of human APC full-length protein. MT-microtubulebinding, DLG-Discs Large tumor-suppressor protein binding domains.Transgenic flies expressing full-length (hAPC/FL) and β-catenin bindingdomains (hAPC/CBD) have been reported here. They were expressed usingeither ptc-GAL4 (D, F-J) or ey-GAL4 (K-M). In all the leg discs shown inB-F, anterior is to the left and ventral is up. (B) Anti-Wg staining toshow wildtype pattern of Wg expression in leg discs. Note that Wg isexpressed only in the ventral-anterior compartment. (C) Expressionpattern of dpp-lacZ reporter gene construct, in wildtype leg disc. Notethat dpp-lacZ is expressed predominantly in the dorsal-anteriorcompartment. (D) hAPC/CBD expression results in the activation ofdpp-lacZ in the ventral-anterior compartment. (E) Dll expression patternin a wildtype leg disc as detected by anti-Dll staining (F)hAPC/CBD-induced ectopic Dll expression (arrow). (G) A wildtype leg.(H-J) human APC-induced leg phenotypes. They include branched legs(arrow in H), duplication of claws (arrow in I) and sex-combs (arrow inJ). (K-M) human APC-induced eye phenotypes. Often, the eyes wereprotruding anteriorly due to ectopic ommatidia on the head capsule.However, the total number of ommatidia was often less than normal (K).Human APC also induced duplication of inter-ommatidial bristles (L-M;arrow in M).

[0202]FIG. 2 shows Human APC suppresses the phenotypes induced byelevated levels of β-catenin/Arm and rescues phenotypes associated withmutant dAPC. (A) Expression pattern of 405-GAL4 in wing discs asindicated by lacZ staining. (B) Flu-Δarm/405-GAL4 induced wingphenotype. Note large number of necrotic patches on the wing blade. (C)Rescue of wing phenotype in flies expressing both hAPC/CBD and Flu-Δarm.Expression of hAPC/CBD alone using 405-GAL4 did not induce anyphenotype. (D) The expression pattern of ey-GAL4 in eye discs asindicated by lacZ staining. (E) Expression of Flu-Δarm in developing eyediscs results in severe loss of ommatidia. (F) Co-expression of hAPC/CBDand Flu-Δarm restores normal organization of ommatidia. (G) Theexpression pattern of 405-GAL4 in eye discs as indicated by lacZstaining. This GAL4 was used to express hAPC/FL in differentiatingretinal neurons. (H) Eye of a dAPC^(Q8) heterozygous fly. Note thepresence of pseudopupil, a dark spot in the middle of the eye(asterisk). (I) Eye of a homozygous dAPC^(Q8) fly. This loss-of-functionmutation in dAPC causes degeneration of retinal neurons, which isreflected in the absence of pseudopupil. Also note increase in eyepigmentation. (J) Rescue of eye phenotype in 405-GAL4; hAPC/FLdAPC^(Q8)/dAPC^(Q8) fly. Note normal pigmentation and re-appearance ofpseudopupil.

[0203]FIG. 3 shows Human APC induced changes in the levels ofβ-catenin/Arm in different cell types. The ptc-GAL4 was used to expresshAPC/CBD (B, D, F, H), hAPC/FL (J) and UAS-Cad^(i5) (K). (A, E)Anti-human APC staining of wildtype leg (A) and wing (E) imaginal discsshowing the expression pattern of endogenous dAPC. (B, F) anti-human APCstaining following hAPC/CBD expression in leg (B) and wing (F) discs.(C, G) anti-Arm staining of wildtype leg (C) and wing (G) discs. (D, H,J) anti-Arm staining following hAPC/CBD expression in leg (D) and wing(H) discs and hAPC/FL expression in wing discs (J). Note increasedβ-catenin/Arm levels. This was true for eye-antennal imaginal discs too.(I) Wing disc expressing hAPC/CBD stained with anti-Arm (green) andanti-Wg (red). Higher levels of β-catenin/Arm are seen only inWg-expressing cells. (K) UAS-Cad^(i5) expression induces uniformaccumulation of β-catenin/Arm all along the A/P axis. (F1, G1, H1)Higher magnification images of F, G, H respectively, to show subcellularlocalization of β-catenin/Arm and human APC. In both wild-type cells(G1) and in human APC-expressing cells (H1), β-catenin/Arm ispredominantly localized near the plasma membrane. This has beenconfirmed by double staining with DAPI, which stains DNA (data notshown). Much of the human APC protein is also seen near the plasmamembrane (F1). In all the wing and leg discs shown in this figure,anterior is to the left and ventral is up.

[0204]FIG. 4 shows Effect of GSK-3 β/Sgg activity on the interactionbetween human APC and β-catenin/Arm. (A) The expression pattern ofvg-GAL4 in wing discs as indicated by lacZ staining. This GAL4 driverwas used to express hAPC/CBD (B, D-F) and/or GSK-3 βSgg (C, D, F). Discsin B-D are stained with anti-Arm and E, F for anti-human APC antibodies.(B) hAPC/CBD expression results in the accumulation of β-catenin/Arm,but only in the D/V boundary. Whereas, over-expression of GSK-3 β/Sgg(C) alone or with hAPC/CBD (D) results in the lowering of β-catenin/Armlevels in both D/V and non-D/V boundary cells. (E) hAPC/CBD expressionas driven by vg-GAL4. (F) Wing disc co-expressing both hAPC/CBD andGSK-3 β/Sgg. human APC expression is robust as in E.

[0205]FIG. 5 shows Enhancement of human APC induced eye phenotypes inDrosophila by two independent deletion mutations in the third chromosomeand by the non-steroid anti-inflammatory drug, indomethacin. Eyephenotypes induced by human APC are arranged in the order of severityfrom 1 to 20. Human APC in wildtype background induced phenotypes ofranks 1-9. Expression of human APC in the presence of either Df(3R)by⁶²or l(3L)67E2 or the drug indomethacin showed much enhanced phenotypes(rank 1-20) often resulting eyeless flies. WT—wild type eye.

[0206] The following examples are given by way of illustrations andtherefore should not be construed to limit the scope of the presentinvention.

EXAMPLES Example 1

[0207] Full-length human APC cDNA clone (Kinzler et al., 1991; SEQ IDNo. 1) was released from its vector (pCMV) by BamH1 digestion. The 8.9kb insert was sub-cloned in to the P-element vector, pCaSpeR-UAS (Brandand Perrimon, 1993). The β-catenin binding domain (comprising ofamino-acids from 959 to 1870; SEQ ID NO. 2) was released from a partialcDNA clone of human APC (FB10B; Kinzler et al., 1991) by EcoR1 and Xba1digestion. The 2.7 kb fragment was independently sub-cloned intopCaSpeR-UAS. The N terminal domain of APC (comprising of amino-acidsfrom 1 to 767; SEQ ID NO. 3) was released from full-length cDNA cloneand was subcloned into pCaSpeR-UAS.

Example 2

[0208] All the constructs were injected into w; Δ2-3 Ki embryos carryinga genetic source of transposase (Cooley et al., 1988). 15 independenttransgenic lines for the full-length in construct and 14 for theβ-catenin binding domain were generated. UAS-dAPC1 was generated by YAhmed and E Wieschaus (unpublished) using cDNA clone for dAPC1, thefirst Drosophila homologue of APC. The X-chromosome insertion ofUAS-dAPC1 was mobilized by crossing to a genetic source of transposase(w; Δ2-3 Sb/Δ2-3 TM2).

Example 3

[0209] All the transgenic flies were crossed to various GAL4 drivers toexpress human APC in different tissues. Recombinant chromosomes andcombinations of other UAS lines, different mutations and/or markers weremade by standard genetic techniques. Other UAS lines used were,UAS-Flu-ΔArm (N-terminal 155 residues are deleted in this construct dueto which over-expressed Arm is resistant to GSK-3β-mediated degradation,but can displace endogenous β-catenin/Arm from the adhesion complex;Zecca et al., 1996), UAS-S10 (another activated form of Arm resistant toGSK-3β-mediated degradation due to an internal deletion of N-termninal43-87 residues, but present both in the nucleus and cytoplasm; Pai etal., 1997), UAS-Zw3 (GSK-3β/Sgg; Steitz et al., 1998), UAS-Cad^(i5)(intracellular domain of Drosophila E-cadherin; Sanson et al., 1996),UAS-Dpp (Frasch, 1995) and UAS-activated Tkv (Thick-vein a receptor ofDpp; Nellen et al., 1994). GAL4 strains used were ptc- and en-GAL4(Brand and Perrimon, 1993), dpp-GAL4 (Morimura et al., 1996), ey-GAL4(Hazelett et al., 1998), vg-GAL4 (Simmonds et al., 1995) and 405-GAL4(LSS, unpublished). Choice of GAL4 drivers for the expression of humanAPC and/or other proteins was mainly determined by the severity of thephenotype.

Example 4

[0210] RNA in situ was done essentially as described by Tautz andPfeifle (1989) using FB10B cDNA clone of human APC as the probe. X-galstaining and immuno-histochemical staining were essentially as described(Ghysen and O'Kane, 1989; Patelet al., 1989). The lacZ reporter geneconstructs used are UAS-lacZ (Brand and Perrimon, 1993) and dpp-lacZ(Blackman et al., 1991). The primary antibodies used are, anti-human APC(Hayashi et al., 1997), anti-Arm (Riggleman et al., 1990), anti-Dll(Vachon et al., 1992) and anti-Wg (Brook and Cohen, 1996). Anti-Arm andanti-Wg were obtained from the Development Studies Hybridoma Bank,University of Iowa, USA.

Example 5

[0211] The adult appendages were processed for microscopy as describedbefore (Shashidhara et al., 1999). For Scanning Electron microscopy(SEM), adult heads were air dried and coated with gold. SEM was carriedout on Hitachi S520 machine.

Example 6

[0212] To examine the effect of lowering endogenous β-catenin/Arm levelson hAPC induced phenotypes, arm⁴ allele of arts(http://flybase.bio.indiana.edu) was used along with hAPC/CBD, whereinflies carrying one copy of hAPC/CBD and one copy of arm⁴ allele allelewere crossed to ptc- and dpp-GAL4 drivers. To rescue mutant phenotypesof dAPC, a III chromosome insertion of UAS-hAPC/FL transgene wascombined with dAPC^(Q8) (Ahmed et al., 1998) and the resultant flieswere crossed to different GAL4 strains, which were also heterozygous fordAPC^(Q8). Homozygous flies were identified with the help of ebonymarker associated with dAPC^(Q8).

Example 7

[0213] To screen for genetic modifiers of human APC function, ey-GAL4was first crossed to a collection of Drosophila mutants (pointmutations, deletions and P-lethal insertions) and then to UAS-hAPC/CBD.Following mutant stocks were used. arm⁴, P-1783, P-1595, P-1659, P-0237,P-1568, Df(3L)AC1, Df(3L) 66C-G28, Df(3L) R-G7, Df(3L) 29A6, Df(3L) Cat,Df(3L) fz GF3b, Df(3L) fz M21, Df(3L) Ly, Df(3L) HR119, Df(3) GN24,Df(3R) Scr, Df(3R) by 10, Df(3R) crb87-4, Df(3R) crb-87-5, Df(3) by 62,Df(3R) p-XT103, Df(3R) Cha7, Df(3R) red¹, Df(3R) C4, Df(3R) M-Kx1,Df(3R) B81, In(3LR) 270, In(3LR) 268 and In(3R) hb^(D1). All theabove-mentioned stocks are described in httD://flybase.bio.indiana.edu.Alleles of l (3)67E2 were generated in the laboratory (R Bajpai,unpublished).

Example 8

[0214] For drug screening, drugs (at different concentrations) wereadded to fly food at <50° C. and left over-night for drying. Care wastaken to ensure homogenous mixing of the drug in the fly food.Blind-tests were carried out, wherein the vials with or without drugswere coded (with the help of other members of the lab). The crosses werealways set-up in triplicates. The method of administering drugsdescribed above is only an example and should not be limited to it asthere can be other ways of administering drugs.

Example 9

[0215] A method for determining the differential expression of genesfollowing mis-expression of human APC in Drosophila. Briefly, RNAsamples would be isolated from tissues expressing and not expressinghuman APC. They would be labeled by RT-PCR and resolved by gelelectrophoresis or labeled probes would be hybridized to arrayed cDNAclones. This would allow us to identify genes that are differentiallyregulated by the expression of human APC. Such RNA-based approach ofscanning the entire Drosophila genome would help identification ofadditional components of Wnt signal transduction pathway, which wouldprovide suitable targets for drug screening against cancer and otherconditions caused by over-activation of β-catenin.

Example 10

[0216] A method for determining the modification and differentialexpression of proteins following mis-expression of human APC inDrosophila. Briefly, proteins would be isolated from tissues expressingor not expressing human APC and would be subjected to 2-D gelelectrophoreses. The protein bands, which show differential mobilitywould be isolated and subjected to further analyses that includes MASSSPEC analyses and protein sequencing.

LIST OF PRIOR ART REFERENCES

[0217] 1. Ahmed, Y., Hayashi, S., Levine, A. and Wieschaus, E. (1998)Regulation of armadillo by a Drosophila APC inhibits neuronal apoptosisduring retinal development. Cell 93, 1171-1182.

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1. A transgenic Drosophila whose genome comprises the full-length humancolon cancer gene Adenomatous Polyposis Coli (APC) having SEQ ID NO.1wherein: (a) said genomic alteration allows mis-expression offull-length human APC in flies in regulated manner, (b) saidmis-expression of the full-length human APC results in developmentalabnormalities, (c) said developmental abnormalities induced by themis-expression of full-length human APC in flies are similar to thoseexhibited by flies carrying mutations in Drosophila wingless gene, and(d) to use the same as an assay system for screening and validatingefficacy of drugs.
 2. The transgenic Drosophila as claimed in claim 1wherein, its genome includes β-catenin binding domain comprising ofamino-acids from 959 to 1870 of SEQ ID NO. 2 from the full length humanAPC gene of SEQ ID NO.1, and this engineered disruption of human APCcomprises only the five of the seven β-catenin binding domains wherein:(a) said genomic alteration allows mis-expression of a truncated versionof human APC in flies in a regulated manner, (b) said mis-expression ofthe said gene construct results in the developmental abnormalities, (c)said developmental abnormalities induced by the mis-expression of thesaid gene construct in flies is similar to those exhibited by fliescarrying mutations in Drosophila wingless gene, (d) said mis-expressionof the said novel construct in regulated manner results in a more severedevelopmental phenotype, and (e) to use the same as an assay system forscreening and validating efficacy of drugs.
 3. The transgenic Drosophilaas claimed in claim 1 wherein, the N terminal domain of APC with aminoacids from 1 to 767 having SEQ ID NO. 3, from the full length human APCgene of SEQ ID NO.1 ,wherein: (a) said genomic alteration allowsmis-expression of human APC in flies in a regulated manner, (b) saidmis-expression of the said novel construct in a regulated mannerresulting in severe abnormalities in fly development duringmetamorphosis, and (c) to use the same as an assay system for screeningand validating efficacy of drugs.
 4. A method for selecting a compoundfor pharmacological activity, which potentially inhibits or enhances thedevelopmental abnormalities induced by the expression of full length andprotein domains of human APC in Drosophila, said method comprising: (a)providing the first, second, and third transgenic fly of claims 1, 2 and3 respectively, wherein said flies have said developmentalabnormalities, (b) administering the said compounds to the saidtransgenic Drosophila at different concentrations, and (c) screening forthe changes in the severity of the phenotype.
 5. A method of determiningvarious Drosophila proteins interacting with full-length and proteindomains human APC protein wherein, said method comprising: (a) providingthe first, second, and third transgenic fly of claims 1, 2 and 3respectively, wherein said flies have said developmental abnormalities,(b) crossing the said transgenic flies individually to a set ofDrosophila strains each of which carries mutation in a different gene orset of genes, and (c) Screening for the change in the severity of thephenotype.
 6. A method for determining the modulation and differentialexpression of genes following the mis-expression of full-length and itsprotein domains human APC in Drosophila wherein, said method comprising:(a) providing the transgenic Drosophila as claimed in claims 1, 2 and 3wherein, the flies have developmental abnormalities, (b) screening fordifferential gene expression using differential display-RT PCR ormicroarray techniques, and (c) identifying genes that are differentiallyregulated on expression of human APC.
 7. A method for determining themodulation and differential expression of proteins following themis-expression of full-length and its protein domain human APC inDrosophila wherein, said method comprising: (a) providing the transgenicDrosophila , as claimed in claims 1, 2 and 3 wherein, the flies havedevelopmental abnormalities, (b) identifying differential geneexpression and protein modifications using proteomics techniques, and(c) identifying gene products that are differentially regulated onexpression of human APC.
 8. A method to study Wnt/Wg signaling inDrosophila said method comprising; (a) providing the transgenicDrosophila, as claimed in claims 1, 2 and 3, (b) crossing thesetransgenic flies to a number of GAL4 drivers to induce targetedexpression of said constructs in various tissues and at differentdevelopmental stages, and (c) examining developmental abnormalities. 9.Methods as claimed in claims 6-8 wherein, examination of developmentalabnormalities using gain-of-function genetic model for human APC tostudy mechanism of various developmental processes such as wing, leg,eye, antennae, and adult cuticle development.
 10. A Method as claimed inclaim 4 wherein, screening and validating efficacy of preventive andtherapeutic drugs following APC gene mis-expression.
 11. A Method asclaimed in claim 4 wherein, human APC pathway is identified using drugselected from a group of compunds comprising anti inflammatory,Analgesics, Antipyretics, and Antineoplastics.
 12. A method as claimedin claim 4 wherein, concentration of said drugs ranging between 50 to500 g/ml of fly food.
 13. Methods as claimed claims 6-8 wherein,examination of developmental abnormalities using gain-of-functiongenetic model for human APC which has advantages to study the DrosophilaWnt/Wg signaling pathway.
 14. A Method as claimed in claim 8 wherein,studying the kinetics of Wnt/Wg signaling during various developmentalstages and in different tissues.
 15. A Method as claimed in claims 5 and7 wherein, new target proteins interacting with β-catenin areidentified.
 16. A Method as claimed in claim 6 wherein, genesinteracting with APC are identified.
 17. Methods as claimed in claims5-8 wherein, examination of developmental abnormalities usinggain-of-fuction genetic model for human APC to study biochemicalfunction of human APC function.
 18. Methods as claimed in claims 5-8wherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC to identify additionalcomponents of Drosophila Wnt/Wg signaling pathway.
 19. A transgenicDrosophila whose genome comprises the full-length human colon cancergene Adenomatous Polyposis Coli (APC) having SEQ ID NO.1 wherein: (a)said genomic alteration allows mis-expression of full-length human APCin flies in regulated manner, (b) said mis-expression of the full-lengthhuman APC results in developmental abnormalities, (c) said developmentalabnormalities induced by the mis-expression of full-length human APC inflies are similar to those exhibited by flies carrying mutations inDrosophila wingless gene, and (d) to use the same as an assay system forscreening and validating efficacy of anti-cancer drugs.
 20. Thetransgenic Drosophila as claimed in claim 19 wherein, its genomeincludes β-catenin binding domain comprising of amino-acids from 959 to1870 of SEQ ID NO. 2 from the full length human APC gene of SEQ ID NO.1,and this engineered disruption of human APC comprises only the five ofthe seven catenin binding domains wherein: (a) said genomic alterationallows mis-expression of a truncated version of human APC in flies in aregulated manner, (b) the mis-expression of the said gene constructresults in the developmental abnormalities, (c) the developmentalabnormalities induced by the mis-expression of the said gene constructin flies is similar to those exhibited by flies carrying mutations inDrosophila wingless gene, (d) mis-expression of the said novel constructin regulated manner results in a more severe developmental phenotype,and (e) to use the same as an assay system for screening and validatingefficacy of anti-cancer drugs.
 21. The transgenic Drosophila as claimedin claim 19 wherein, the N terminal domain of APC with amino acids from1 to 767 having SEQ ID NO. 3, from the full length human APC gene of SEQID NO.1 wherein: (a) the said genomic alteration allows mis-expressionof human APC in flies in a regulated manner, (b) the mis-expression ofthe said novel construct in a regulated manner resulting in severeabnormalities in fly development during metamorphosis, and (c) to usethe same as an assay system for screening and validating efficacy ofanti-cancer drugs.
 22. A method for selecting a compound for anti-canceractivity, which potentially inhibits or enhances the developmentalabnormalities induced by the expression of full length and proteindomains of human APC in Drosophila, said method comprising: (a)providing the first, second, and third transgenic fly of claims 19, 20,and 21 respectively, wherein said flies have said developmentalabnormalities, (b) administering the said compounds to the saidtransgenic Drosophila at different concentrations, and (c) screening forthe change in the severity of the phenotype.
 23. A method of determiningvarious Drosophila proteins interacting with full-length and proteindomains human APC protein wherein, said method comprising: (a) providingthe first, second, and third transgenic fly of claims 19, 20, and 21respectively, wherein said flies have said developmental abnormalities,(b) crossing the said transgenic flies individually to a set ofDrosophila strains each of which carries mutation in a different gene orset of genes, and (c) Screening for the change in the severity of thephenotype.
 24. A method for determining the modulation and differentialexpression of genes following the mis-expression of full-length and itsprotein domains human APC in Drosophila wherein, said method comprising:(a) providing the transgenic Drosophila as claimed in claims 19, 20, and21 wherein, the flies have developmental abnormalities, (b) screeningfor differential gene expression using differential display-RT PCR ormicroarray techniques, and (c) identifying genes that are differentiallyregulated on expression of human APC.
 25. A method for determining themodulation and differential expression of proteins following themis-expression of full-length and its protein domain human APC inDrosophila wherein, said method comprising: (a) providing the transgenicDrosophila , as claimed in claims 19, 20, and 21 wherein, the flies havedevelopmental abnormalities, (b) identifying differential geneexpression and protein modifications using proteomics techniques, and(c) identifying gene products that are differentially regulated onexpression of human APC.
 26. A method to study Wnt/Wg signaling inDrosophila said method comprising; (a) providing the transgenicDrosophila, as claimed in claims 19-21, (b) crossing these transgenicflies to a number of GAL4 drivers to induce targeted expression of saidconstructs in various tissues and at different developmental stages, and(c) examining developmental abnormalities.
 27. Methods as claimed inclaims 24-26 wherein, examination of developmental abnormalities usinggain-of-function genetic model for human APC to study mechanism ofvarious developmental processes such wing, leg, eye, antennae, and adultcuticle development.
 28. A Method as claimed in claim 22 wherein,screening and validating efficacy of anti-cancer drugs following APCgene mis-expression.
 29. A Method as claimed in claim 22 wherein, humanAPC pathway is identified using drugs selected from a group of compoundscomprising anti inflammatory, Analgesics, Antipyretics, andAntineoplastics.
 30. A method as claimed in claim 22 wherein,concentration of said anti-cancer drugs ranging between 50 to 500 μg/mlof fly food.
 31. Methods as claimed claims 24-26 wherein, examination ofdevelopmental abnormalities using gain-of-function genetic model forhuman APC which has advantages to study the Drosophila Wnt/Wg signalingpathway.
 32. A Method as claimed in claim 26 wherein, studying thekinetics of Wnt/Wg signaling during various developmental stages and indifferent tissues.
 33. Methods as claimed in claims 23 and 25 wherein,new target proteins interacting with β-catenin are identified.
 34. AMethod as claimed in claim 24 wherein, genes interacting with APC areidentified.
 35. Methods as claimed in claims 23-26 wherein, examinationof developmental abnormalities using gain-of-function genetic model forhuman APC to study biochemical function of human APC function. 36.Methods as claimed in claims 23-26 wherein, examination of developmentalabnormalities using gain-of-function genetic model for human APC toidentify additional components of Drosophila Wnt/Wg signaling pathway.